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Characterization of ClC‐2 Chloride Channel/PKA Interaction in Mouse Heart
Author(s) -
Huang Zheng Maggie,
Britton Fiona,
Yuan Can,
An Changlong,
Hatton William,
Duan Dayue
Publication year - 2008
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.22.1_supplement.721.5
Subject(s) - phosphorylation , chloride channel , protein kinase a , dephosphorylation , chemistry , immunoprecipitation , protein subunit , western blot , patch clamp , xenopus , microbiology and biotechnology , receptor , biology , biochemistry , phosphatase , gene
Background: A ClC‐2 encoded chloride inward rectifier channel (Cl.ir) has been identified in the heart of several species. Cl.ir may play a role in regulation of pacemaker activity of the heart. In this study, we studied the regulation of mouse cardiac ClC‐2 channels by protein kinase A (PKA) phosphorylation. Methods: RT‐PCR and Western Blot were used to examine the distribution of mClC‐2 in mouse heart. Whole‐cell voltage‐clamp was used to study the regulation mClC‐2 in NIH/3T3 cells. Coimmunoprecipitation (CoIP) and yeast two hybrid (Y2H) were used to identify the binding partners of mClC‐2 in the heart. Results: 1). ClC‐2 exists in mouse heart. Analysis showed 765Ser as a PKA phosphorylation site. 2). Expression of ClC‐2 in NIH/3T3 cells generated functional channels. Treatment by 8‐Br‐cAMP to activate PKA increased current density from −16.2±2.8 to −20.6±3.5 pA/pF at −140mV by 27.4±5% (mean±SD, n=3). The increase was reversed to −15.5±3.3pA/pF by 26.6±4.1% upon PKA inhibition. Phosphotases PP1 and PP2 inhibitor Calyculin A further increased current by 22±3.1%. 3). CoIP using mouse heart and mouse atrial cell line HL1 lysates both showed association of ClC‐2 to 1 adrenergic receptors, PKA catalytic subunit, and PP1. 4). Y2H identified PP1 as a binding partner of ClC‐2. Conclusion: Mouse cardiac ClC‐2 channels may be regulated by phosphorylation/dephosphorylation through a direct interaction with PKA and PP1. Source of support: National Institutes of Health, National Center for Research Resources, National Heart, Lung, and Blood Institute and American Heart Association Western Affiliate.